Two proteins have been identified in human erythrocyte membranes which regulate complement activation at the cell surface. Although it has been recognized for some time that humoral proteins are important regulators of complement activation, it has only recently been appreciated that integral membrane proteins have a distinct and essential role in the regulatory process. For example, the abnormal erythrocytes of paroxysmal nocturnal hemoglobinuria, which are lysed by complement soon after entering the blood stream, were shown to lack one of the membrane regulatory proteins (DAF). Other experiments have shown that the complement regulatory functions of DAF, but not of the C3b receptor (CR1) are necessary to prevent complement activation by human erythrocytes. The proposed project seeks to elucidate the role that membrane-associated proteins play in regulating complement activation and in processing particles which activate the human complement system. The research will utilize purified DAF and CR1 where appropriate but will emphasize the measurement of regulatory activities expressed by DAF and CR1 in cell membranes. DAF purified from erythrocytes will be characterized in terms of its structural, biochemical and complement regulatory functions. Assays have been developed to quantitate the regulatory functions of DAF and CR1 in intact membranes and these will be used to compare activities of the purified proteins with their in situ activities on various cells. DAF has been identified on platelets and lymphocytes and its distribution and function on other peripheral blood cells, endothelial cells and cells from other tissues will be delineated. The research will assess the role of DAF in the CR1-mediated processing of immune complexes and of particles which activate the alternative pathway. Complement enzymes on these particles can be bound to the surface of many cells via Fc or complement receptors. The roles of the regulatory proteins DAF and CR1 in protecting such cells from complement-mediated damage by these enzymes will be examined using polyclonal and monoclonal antibodies to inhibit specific functions. The results of these studies should provide insights into how complement-activating particles are processed and how defects in the membrane proteins might be expressed in disease.